Biology subjects

Lee, K. M.

Publications and source records attributed to Lee, K. M..

16 recordsLinked to original sources

bioRxiv2025

Hunchback functions in the post-mitotic larval MDN to restrict axon outgrowth, synapse formation, and backward locomotion

During neurodevelopment, a single progenitor cell can generate many different neuron types. As these neurons mature, they form unique morphologies, integrate into neural circuits, and contribute to behavior. However, the integration of these developmental events is understudied. Here, we show that the same transcription factor is important for both the generation of neuronal diversity and maintaining mature neuronal identity, providing novel insights on how the generation of neuronal identity and morphology are coordinated. We utilized a previously characterized larval locomotor circuit in Drosophila, where activation of the Moonwalker Descending Neuron (MDN) triggers backward locomotion via its presynaptic connection with the premotor neuron A18b. MDN expresses the temporal transcription factor Hunchback (Hb), which has a well-characterized role in neural progenitors. Loss of Hb in the post-mitotic MDN increases axon/dendrite branching, leading to additional functional synapses on A18b and increasing backward locomotion. We conclude that the endogenous function of Hb is to restrain axon/dendrite outgrowth, including limiting MDN-A18b synapses, thereby dampening backward locomotion. Our work provides insights on how a transcription factor can have different functions throughout life - i.e. Hb generates neuronal diversity in the progenitor and regulates neuronal connectivity in the mature neuron to generate an appropriately tuned behavior.

neuroscience↗

bioRxiv2025

Single-molecule analysis of gap and nick binding by LIG1 and LIG3α at the final step of DNA repair

DNA ligase (LIG) 1 and LIG3 repair broken single-strand breaks in the phosphodiester backbone at the final ligation step of DNA excision repair pathways, and complement each other during nuclear replication in case of unligated Okazaki fragments. We previously reported that both ligases discriminate against nicks containing non-canonical ends and ligate gap intermediate if left unfilled by DNA polymerases. However, it remains unknown how the dynamics of DNA binding differ for gap versus nick substrates by LIG1 and LIG3 at single-molecule level. Here, using total internal reflection fluorescence (TIRF) and ligation assays, we showed that LIG3 binds less frequently but forms longer-lived complex than LIG1 for nicks containing canonical A:T, mismatch G:T, and damaged 8oxoG:A, and they exhibit subtle differences in discriminating unusual ends. Moreover, our results identified gap DNA as a new target to which LIG1 and LIG3 can bind as efficient as their preferential nick sites. We showed gap ligation and observed that more percentage of LIG1 molecules form stable long-lived complex on DNA containing one nucleotide gap, whereas LIG3 forms short-lived gap complex without any differences in the percentage of molecule forming gap-bound complex. Finally, our findings demonstrated that LIG1 can still stably bind to larger gaps with better recognition, whereas LIG3 binding becomes further infrequent and shorter-lived. Overall, our study provides single-molecule insights into intricate differences between LIG1 and LIG3 for binding to a range of mutagenic and deleterious DNA repair and replication intermediates that could be a threat for maintaining genome stability at the final step.

biochemistry↗

bioRxiv2025

Characterization of nick binding and sealing by LIG1 Huntington's disease-asssociated K845N variant at biochemical, structural, and single-molecule levels

DNA ligase 1 (LIG1) joins broken strand breaks and discriminates against nicks containing mismatch or oxidative damage. Huntingtons disease (HD)-associated mutation K845N in LIG1 gene has been predicted to be onset delaying and suppresses CAG repeat expansion. Yet, how this mutation impacts faithful nick sealing and efficient DNA binding by LIG1 remains unknown. Here, using biochemistry, X-ray crystallography, and total internal reflection fluorescence microscopy, we comprehensively characterized the impact of LIG1 HD-associated mutation at biochemical, structural, and single-molecule levels. Our results showed a reduced ligation efficiency by LIG1 K845N variant in the presence of nick substrates containing all possible 12 mismatches, 8-oxoG, and ribonucleotides at the 3-end when compared with the wild-type enzyme. Furthermore, our structures provided an atomic insight into differences in distances between the functional groups of K/N845 and DNA ends, demonstrating similar conformation and a lack of large scale alternations at the ligase active site. Finally, our single-molecule measurements in real-time revealed that K845N mutant binds less frequently for shorter life-time to nick DNA than wild-type protein. Overall findings contribute to understand the mechanism by which LIG1 ensures fidelity and nick binding to maintain genome stability at the final ligation step in normal versus disease states.

biochemistry↗

bioRxiv2025

Contrasting genetic differentiation of urban and rural populations of two grassland lepidopterans across Europe

Urbanisation is transforming environments globally. The altered abiotic conditions and biotic interactions in urban habitats impose divergent selection pressures on urban versus rural populations, while genetic drift may also be significant in typically small urban populations. A key question in urban evolution concerns the origin and spread of urban genotypes. Examples exist of both single and multiple origins of urban genotypes, but these have proven difficult to generalize. Here, we address genetic differentiation among urban populations, among rural populations, and between urban and rural populations. We conducted an extensive population genomic double digest restriction-site associated DNA sequencing analysis of two non-model grassland lepidopterans, Coenonympha pamphilus and Chiasmia clathrata, across Europe. The genetic population structures of the study species were strikingly different: Co. pamphilus showed strong population differentiation, while this was almost absent in Ch. clathrata, which instead showed signs of high current and past gene flow among populations. Results of Co. pamphilus are consistent with multiple origins of urban populations, and multiple origins seem plausible also in Ch. clathrata. These results suggest that past and large-scale population dynamics need to be integrated into urban evolution research, because population history affects urban evolutionary dynamics.

evolutionary biology↗

bioRxiv2024

Eosinophils promote monocyte to macrophage differentiation and anti-bacterial immunity

Chemokine receptors control cell migration within the body. Here we reveal a novel interaction between eosinophils and monocytes in the bone marrow, indirectly controlled by the atypical chemokine receptor ACKR2. We demonstrate that ACKR2 maintains eosinophil levels within the bone marrow by scavenging CCL11. In the absence of ACKR2, elevated CCL11 leads to increased egress of eosinophils from the bone marrow into the bloodstream. As a result, eosinophil and monocyte interactions are reduced within the bone marrow niche, leading to changes in monocyte gene expression. Monocytes from ACKR2-/- mice are recruited to the tissues but are fundamentally altered in their ability to differentiate into macrophages, in the lung, peritoneal cavity and cavity wall. Bacterial elimination is impaired in ACKR2-/- mice during peritoneal infection. ACKR2 is therefore a key regulator of eosinophil-driven monocyte education in the bone marrow, required for full monocyte differentiation and macrophage function within the tissues.

immunology↗

bioRxiv2024

Biochemical, structural, and single-molecule characterization of LIG1 active site mutants demonstrate role of F635 and F872 residues for faithful ligation

Human DNA ligase 1 (LIG1) finalizes DNA repair pathways by an ultimate ligation step and discriminates against nicks containing unusual ends, yet the contribution of the conserved active site residues for faithful end joining remains unknown. Here, using biochemistry, X-ray crystallography, and single-molecule approaches, we comprehensively characterized LIG1 mutants carrying Ala(A) and Leu(L) substitutions at the active site residues Phe(F)635 and Phe(F)872. Our results showed an abolished ligation of nick DNA substrates with all 12 non-canonical mismatches, while the mutagenic nick sealing of oxidatively damaged ends by wild-type enzyme is significantly reduced by F635A/L and F872A/L substitutions. Furthermore, sugar discrimination against a single ribonucleotide at 3- or 5-end of nick DNA is distinctly affected depending on architecture of 3-terminus:template base pairing. Finally, our LIG1 structures demonstrated the importance of DNA end alignment governed by the distance to nick site through F635 and F872 residues, and single-molecule measurements showed similar nick DNA binding modes for LIG1 wild-type and active site mutants in real-time. Overall, our study provides a mechanistic insight into the mechanism by which conserved F635 and F872 residues contribute to ligation efficiency of nick repair intermediates that mimic DNA polymerase-mediated mismatch, damaged, or ribonucleotide insertion products and how LIG1 ensures faithful end joining at the final step of DNA repair to maintain genome integrity.

biochemistry↗

bioRxiv2024

Mutagenic ligation of polβ mismatch insertion products during 8-oxoG bypass by LIG1 and LIG3α at the downstream steps of base excision repair pathway

Base excision repair (BER) maintains genome integrity by fixing oxidized bases that could be formed when reactive oxygen species attack directly on the DNA. We previously reported the importance of a proper coordination at the downstream steps involving gap filling by DNA polymerase (pol) {beta} and subsequent nick sealing by DNA ligase (LIG) 1 or 3. Yet, how the fidelity of 8-oxoG bypass by pol{beta} affects the efficiency of ligation remains unclear. Here, we show that LIG1 can seal nick products of pol{beta} after both dATP and dCTP insertions during 8- oxoG bypass, while ribonucleotide insertions completely diminish the repair coordination with both ligases, highlighting a critical role for nucleotide selectivity in maintaining BER accuracy. Furthermore, our results demonstrate that LIG3 exhibits an inability to ligate nicks of pol{beta} dCTP:8-oxoG insertion or with preinserted 3-dC:8-oxoG. Finally, AP-Endonuclease 1 (APE1) proofreads nick repair intermediates containing 3-dA/rA and 3-dC/rC mismatches templating 8-oxoG. Overall, our findings provide a mechanistic insight into how the dual coding potential of the oxidative lesion and identity of BER ligase govern mutagenic versus error-free repair outcomes at the final steps and how the ribonucleotide challenge compromises the BER coordination leading to the formation of deleterious repair intermediates.

biochemistry↗

bioRxiv2024

TRPV4 is expressed by enteric glia and muscularis macrophages of the colon but does not play a prominent role in colonic motility

BackgroundMechanosensation is an important trigger of physiological processes in the gastrointestinal tract. Aberrant responses to mechanical input are associated with digestive disorders, including visceral hypersensitivity. Transient Receptor Potential Vanilloid 4 (TRPV4) is a mechanosensory ion channel with proposed roles in visceral afferent signaling, intestinal inflammation, and gut motility. While TRPV4 is a potential therapeutic target for digestive disease, current mechanistic understanding of how TRPV4 may influence gut function is limited by inconsistent reports of TRPV4 expression and distribution. MethodsIn this study we profiled functional expression of TRPV4 using Ca2+ imaging of wholemount preparations of the mouse, monkey, and human intestine in combination with immunofluorescent labeling for established cellular markers. The involvement of TRPV4 in colonic motility was assessed in vitro using videomapping and contraction assays. ResultsThe TRPV4 agonist GSK1016790A evoked Ca2+ signaling in muscularis macrophages, enteric glia, and endothelial cells. TRPV4 specificity was confirmed using TRPV4 KO mouse tissue or antagonist pre-treatment. Calcium responses were not detected in other cell types required for neuromuscular signaling including enteric neurons, interstitial cells of Cajal, PDGFR+ cells, and intestinal smooth muscle. TRPV4 activation led to rapid Ca2+ responses by a subpopulation of glial cells, followed by sustained Ca2+ signaling throughout the enteric glial network. Propagation of these waves was suppressed by inhibition of gap junctions or Ca2+ release from intracellular stores. Coordinated glial signaling in response to GSK1016790A was also disrupted in acute TNBS colitis. The involvement of TRPV4 in the initiation and propagation of colonic motility patterns was examined in vitro. ConclusionsWe reveal a previously unappreciated role for TRPV4 in the initiation of distension-evoked colonic motility. These observations provide new insights into the functional role of TRPV4 activation in the gut, with important implications for how TRPV4 may influence critical processes including inflammatory signaling and motility. SummaryO_LITRPV4 is expressed by equivalent cell types in the rodent and primate (monkey and human) colon. This mechanosensitive ion channel has proposed roles in inflammation, visceral afferent signaling, and colonic motility. C_LIO_LINew analysis methods were developed to examine cellular communication in the enteric glial network. This approach revealed new insights into inflammation-associated changes in glial connectivity. C_LIO_LINew roles for TRPV4 in transduction of distension-evoked responses in the colon and colonic motility were identified. C_LI Key findingsWe have defined the cell types that functionally express TRPV4 in the gut wall. These include enteric glia, endothelia of blood and lymphatic vessels, mMac, and extrinsic afferent nerves. TRPV4- dependent Ca2+ signaling was not detected in enteric neurons, PDGFR cells, interstitial cells of Cajal and smooth muscle cells, which are important drivers of gut motility. These observations align with our experimental evidence for limited involvement of TRPV4 in neuromuscular transmission and propagating colonic motility. New and NoteworthyO_LINovel cellular sites of functional TRPV4 expression in the GI tract were identified and compared across multiple vertebrate species. New analytical approaches to characterize enteric glial communication in a spatiotemporal manner were developed. C_LIO_LIA supporting role for TRPV4 in the initiation of propagating colonic contractions in response to distension was demonstrated. Potential mechanisms that contribute to TRPV4-mediated effects on GI function were identified. C_LIO_LITRPV4-dependent activity in enteric glia is enhanced in inflammation, consistent with current evidence for inflammation-associated sensitization of TRPV4 on visceral afferents and a major role in mechanically evoked nociceptive signaling. C_LIO_LIPair correlation analysis was used to examine spatial connectivity of Ca2+ signaling, enabling demonstration of dysregulated glial communication in acute inflammation. C_LI

physiology↗

bioRxiv2023

Cortical and subcortical mapping of the allostatic-interoceptive system in the human brain: replication and extension with 7 Tesla fMRI

The brain continuously anticipates the energetic needs of the body and prepares to meet those needs before they arise, called allostasis. In support of allostasis, the brain continually models the sensory state of the body, called interoception. We replicated and extended a large-scale system supporting allostasis and interoception in the human brain using ultra-high precision 7 Tesla functional magnetic resonance imaging (fMRI) (N = 90), improving the precision of subgenual and pregenual anterior cingulate topography combined with extensive brainstem nuclei mapping. We observed over 90% of the anatomical connections published in tract-tracing studies in non-human animals. The system also included regions of dense intrinsic connectivity broadly throughout the system, some of which were identified previously as part of the backbone of neural communication across the brain. These results strengthen previous evidence for a whole-brain system supporting the modeling and regulation of the internal milieu of the body.

neuroscience↗

bioRxiv2021

Hunchback activates Bicoid in post-mitotic Pair1 neurons to regulate synapse number

The proper formation and function of neural circuits is crucial for cognition, sensation, and behavior. Neural circuits are highly-specific, and this specificity is dependent on neurons developing key features of their individual identities: morphology, anatomical location, molecular expression and biophysiological properties. Previous research has demonstrated that a neurons identity is, in part, generated by the temporal transcription window the neuron is born in, and the homeodomain transcription factors expressed in the mature neuron. However, whether temporal transcription factors and homeodomain transcription factors regulate neural circuit formation, maintenance and function remains unknown. Here, we utilize a well-characterized neural circuit in the Drosophila larvae, the Pair1 neuron. We determined that in the Pair1 neuron, the temporal transcription factor Hunchback activates the homeodomain transcription factor Bicoid (Bcd). Both Hunchback and Bcd are expressed in Pair1 throughout larval development. Interestingly, Hunchback and Bcd were not required in Pair1 for neurotransmitter identity or axonal morphology, but were required for synapse density. We found that these transcription factors were functioning post-mitotically in Pair1 to regulate synapse density. Additionally, knocking down Hunchback and Bcd in Pair1 neurons disrupted the behavioral output of the circuit. We utilized the genetic tool TransTango to determine that Hunchback function in Pair1 is to repress forming synapses with erroneous neurons. To our knowledge, these data are the first to show Hunchback activating Bcd expression, as well as the first to demonstrate a role for Hunchback and Bcd post-mitotically.

neuroscience↗

bioRxiv2020

The timing of human adaptation from Neanderthal introgression

Admixture has the potential to facilitate adaptation by providing alleles that are immediately adaptive in a new environment or by simply increasing the long term reservoir of genetic diversity for future adaptation. A growing number of cases of adaptive introgression are being identified in species across the tree of life, however the timing of selection, and therefore the importance of the different evolutionary roles of admixture, is typically unknown. Here, we investigate the spatio-temporal history of selection favoring Neanderthal-introgressed alleles in modern human populations. Using both ancient and present-day samples of modern humans, we integrate the known demographic history of populations, namely population divergence and migration, with tests for selection. We model how a sweep placed along different branches of an admixture graph acts to modify the variance and covariance in neutral allele frequencies among populations at linked loci. Using a method based on this model of allele frequencies, we study previously identified cases of Neanderthal adaptive introgression. From these, we identify cases in which Neanderthal introgressed alleles were quickly beneficial and other cases in which they persisted at low frequency for some time. For some of the alleles that persisted at low frequency, we show that selection likely independently favored them later on in geographically separated populations. Our work highlights how admixture with ancient hominins has contributed to modern human adaptation and contextualizes observed levels of Neanderthal ancestry in present-day and ancient samples.

evolutionary biology↗

bioRxiv2019

Parallel and nonparallel genomic responses contribute to herbicide resistance in Ipomoea purpurea, a common agricultural weed

The repeated evolution of herbicide resistance has been cited as an example of genetic parallelism, wherein separate species or genetic lineages utilize the same genetic solution in response to selection. However, most studies that investigate the genetic basis of herbicide resistance examine the potential for changes in the protein targeted by the herbicide rather than considering genome-wide changes. We used a population genomics screen and targeted exome re-sequencing to uncover the potential genetic basis of glyphosate resistance in the common morning glory, Ipomoea purpurea, and to determine if genetic parallelism underlies the repeated evolution of resistance across replicate resistant populations. We found no evidence for changes in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosates target protein, that were associated with resistance, and instead identified five genomic regions that show evidence of selection. Within these regions, genes involved in herbicide detoxification--cytochrome P450s, ABC transporters, and glycosyltransferases--are enriched and exhibit signs of selective sweeps. One region under selection shows parallel changes across all assayed resistant populations whereas other regions exhibit signs of divergence. Thus, while it appears likely that the physiological mechanism of resistance in this species is likely the same among resistant populations, we find patterns of both similar and divergent selection across separate resistant populations at particular loci.

evolutionary biology↗

bioRxiv2019

Detection of cross-contamination and strong mitonuclear discordance in two species groups of sawfly genus Empria (Hymenoptera, Tenthredinidae)

In several sawfly taxa strong mitonuclear discordance has been observed, with nuclear genes supporting species assignments based on morphology, whereas the barcode region of the mitochondrial COI gene suggesting different relationships. As previous studies were based on only few nuclear genes, the causes and the degree of mitonuclear discordance remain ambiguous. Here, we obtain genomic-scale ddRAD data together with Sanger sequencing of mitochondrial COI and two to three nuclear protein coding genes to investigate species limits and mitonuclear discordance in two closely related species groups within the sawfly genus Empria. As found previously based on nuclear ITS and mitochondrial COI sequences, species are in most cases supported as monophyletic based on previous and new nuclear data reported here, but not based on mitochondrial COI. This mitonuclear discordance can be explained by occasional mitochondrial introgression with little or no nuclear gene flow, a pattern that might be common in haplodiploid taxa with slowly evolving mitochondrial genomes. Some species in E. immersa group are not recovered as monophyletic also based on nuclear data, but this could partly be because of unresolved taxonomy. Preliminary analyses of ddRAD data did not recover monophyly of E. japonica within E. longicornis group (three Sanger sequenced nuclear genes strongly supported monophyly), but closer examination of the data and additional Sanger sequencing suggested that both specimens were substantially (possibly 10-20% of recovered loci) cross-contaminated. A reason could be due to specimen identification tag jumps during sequencing library preparation of pooled specimens that in previous studies have been shown to affect up to 2.5% of the sequenced reads. We provide an R script to examine patterns of identical loci among the specimens and estimate that cross-contamination rate is not unusually high for our ddRAD dataset as a whole (based on counting identical sequences between immersa and longicornis groups that are well separated from each other and probably do not hybridise). The high rate of cross-contamination for both E. japonica specimens might be explained by small number of recovered loci (~1000) compared to most other specimens (>10 000 in some cases) because of poor sequencing results. We caution drawing unexpected biological conclusions when closely related specimens are pooled before sequencing and tagged only at one end of the molecule or at both ends using unique combination of limited number of tags (less than the number of specimens).

evolutionary biology↗

bioRxiv2018

Population genomics perspectives on convergent adaptation

Convergent adaptation is the independent evolution of similar traits conferring a fitness advantage in two or more lineages. Cases of convergent adaptation inform our ideas about the ecological and molecular basis of adaptation. In judging the degree to which putative cases of convergent adaptation provide independent replication of the process of adaptation, it is necessary to establish the degree to which the evolutionary change is unexpected under null models and to show that selection has repeatedly, independently driven these changes. Here, we discuss the issues that arise from these questions particularly for closely-related populations, where gene flow and standing variation add additional layers of complexity. We outline a conceptual framework to guide intuition as to the extent to which evolutionary change represents the independent gain of information due to selection and show that this is a measure of how surprised we should be by convergence. Additionally, we summarize the ways population and quantitative genetics and genomics may help us address questions related to convergent adaptation, as well as open new questions and avenues of research.

evolutionary biology↗

bioRxiv2018

Generation of four iPSC lines from peripheral blood mononuclear cells (PBMCs) of an Attention Deficit Hyperactivity Disorder (ADHD) individual and a healthy sibling in an Australia-Caucasian family

Peripheral blood mononuclear cells were donated by a male teenager with clinically diagnosed Attention Deficit Hyperactivity Disorder under the Diagnostic and Statistical Manual of Mental Disorders IV criteria and his unaffected male sibling. Induced pluripotent stem cells were developed using integration-free Sendai Reprogramming factors containing OCT, SOX2, KLF4, and c-MYC. All four iPSC lines displayed pluripotent cell morphology, pluripotency-associated factors at protein level, alkaline phosphatase enzymatic activity, male karyotype of 46, XY, and in vitro differentiation capacity into all the three germ layers and negative for Mycoplasma.

neuroscience↗

bioRxiv2017

Distinguishing among modes of convergent adaptation using population genomic data

Geographically separated populations can convergently adapt to the same selection pressure. Convergent evolution at the level of a gene may arise via three distinct modes. The selected alleles can (1) have multiple independent mutational origins, (2) be shared due to shared ancestral standing variation, or (3) spread throughout subpopulations via gene flow. We present a model-based, statistical approach that utilizes genomic data to detect cases of convergent adaptation at the genetic level, identify the loci involved and distinguish among these modes. To understand the impact of convergent positive selection on neutral diversity at linked loci, we make use of the fact that hitchhiking can be modeled as an increase in the variance in neutral allele frequencies around a selected site within a population. We build on coalescent theory to show how shared hitchhiking events between subpopulations act to increase covariance in allele frequencies between subpopulations at loci near the selected site, and extend this theory under different models of migration and selection on the same standing variation. We incorporate this hitchhiking effect into a multivariate normal model of allele frequencies that also accounts for population structure. Based on this theory, we present a composite-likelihood-based approach that utilizes genomic data to identify loci involved in convergence, and distinguishes among alternate modes of convergent adaptation. We illustrate our method on genome-wide polymorphism data from two distinct cases of convergent adaptation. First, we investigate the adaptation for copper toxicity tolerance in two populations of the common yellow monkey flower, Mimulus guttatus. We show that selection has occurred on an allele that has been standing in these populations prior to the onset of copper mining in this region. Lastly, we apply our method to data from four populations of the killifish, Fundulus heteroclitus, that show very rapid convergent adaptation for tolerance to industrial pollutants. Here, we identify a single locus at which both independent mutation events and selection on an allele shared via gene flow, either slightly before or during selection, play a role in adaptation across the species range.

evolutionary biology↗